4 The effects of clodronate disodium on normal and inflamed equine cartilage in vitro

Abstract

Bisphosphonates (BP) are a group of drugs which modulate osteoclastic resorption and alter bone turnover. BP are only approved for use in navicular syndrome in adult horses, however, these drugs are being used off-label for their potential anti-inflammatory and pain-relieving effects. When the BP, clodronate disodium (CLO), is administered intramuscularly at the labeled dose, synovial fluid concentrations are detectable within 2 h of administration. There are currently no published studies evaluating the effects of CLO on equine joint tissues. This study evaluated the effects of CLO on normal and inflamed equine cartilage explants in vitro. Cartilage explants were collected from the femorotibial joints of 3 horses which underwent euthanasia for reasons other than stifle osteoarthritis. Only visually normal cartilage was selected for biopsy. The harvested cartilage from each femorotibial joint was subdivided into 24-well plates. Cartilage explants were subjected to 6 different treatments with 8 replicates each, after a 24 h equilibration period: Control (Con), CLO 5 ng/ml (C/lo), CLO 50 ng/ml (C/hi), rEqIL-1β 10 ng/ml (IL), C/lo plus IL (ILC/lo) and C/hi concentration plus IL (ILC/hi). Explant culture media was collected every 24 h and stored at −20°C until analysis of sulfated glycosaminoglycan (GAG) content and nitric oxide (NO) content using a Dimethylmethylene Blue (DMMB) assay and a Greiss reaction, respectively. At the conclusion of the experiment (96 h), cartilage explants were papain-digested and GAG were quantified using a DMMB assay. Data were analyzed using PROC MIXED procedure (SAS 9.4), where horses were considered a random effect, and treatment and day were fixed effects. For all analyses, values of P < 0.05 were considered significant. At d 1, d 2, and d 3, the IL treatment group had a significantly higher concentration of GAG within the media compared with the Con group ( P < 0.05), indicating the expected catabolic effect of IL-1β on the explants. Treatments with clodronate (C/lo, C/hi) alone did not result in an increase in GAG or NO within the media compared with the control indicating CLO did not have a cytotoxic effect. However, the addition of CLO to the IL-1β treated explants (ILC/lo, ILC/hi) did not result in a significant decrease in GAG or NO release into the media when compared with rEqIL-1β (IL) alone. No differences were observed between treatments when GAG content of cartilage explants was measured following papain-digestion. Our in vitro study suggests that CLO does not result in cartilage toxicity. However, no anti-inflammatory or chondroprotective effects were observed.